1. Technical Field
The present invention generally relates to a non-amphiphile-based water-in-water emulsion composition and uses thereof.
2. Background Information
The use of surfactant materials has been known as early as 2,800 B.C. as evidenced by the discovery of soap-like materials in clay cylinders during the excavation of ancient Babylon (Routh, H. B et al., A. Clin. Dermatol. 1996, 14, (1), 3-6; Hunt, J. A., The Pharm. J. 1999, 263, (7076), 985-989). Since then, the science of emulsions has been believed to depend on the existence of amphiphilic molecules in a solution—where the aliphatic part of the molecule is excluded from water to either self-assemble into different aggregate structures or to solvate hydrophobic molecules into a dispersion of oily materials in water (Routh, H. B et al., A. Clin. Dermatol. 1996, 14, (1), 3-6).
Colloidal and interfacial sciences are rapidly merging with advances in soft and condensed matter physics as well as with biological phenomena at interfaces (Terentjev, E. M., Europhys. Lett. 1995, 32, (7), 607-12; Weitz, D. A., Nature 2001, 410, (6824), 32-33). The impact of liquid crystalline materials on the structure of emulsions is striking for both water-in-oil and oil-in-water emulsions (Tixier, T. et al., Langmuir 2006, 22, (5), 2365-2370). For instance, Poulin and Weitz have reported a linear ordering of water droplets in an anisotropic medium of nematic liquid crystal (Poulin, P. et al., Science 1997, 275, (5307), 1770-1773). This order is believed to arise from a delicate balance between the stabilization of the emulsion by surfactants and the anisotropic forces exerted by the liquid crystals (Poulin, P. et al., Science 1997, 275, (5307), 1770-1773; Drzaic, P. S., Liquid Crystal Dispersions. Wiley-Interscience: Singapore, 1995, Vol. 1; Poulin, P. et al., Phys. Rev. E: Stat. Phys. Plasmas, Fluids, 1998, 57, (1), 626-637). Seminal work on liquid crystal droplets dispersed in water (oil-in-water) was also done by Lavrentovich and coworkers (Lavrentovich, O. D. et al., Phys. Rev. E: Stat. Phys. Plasmas, Fluids, 1998, 57, (6), R6269-R6272; Volovik, G. E. et al., JETP Lett. 1983, 58, 1159-1167). Some applications for nematic liquid crystal droplets dispersed in a polymer solution were also developed for switchable windows and light valves (Doane, J. W. et al., Macromol. Symp. 1995, 96, (International Conference on Liquid Crystal Polymers, 1994), 51-60; Fernandez-Nieves, A. et al., Phys. Rev. Lett. 2004, 92, (10), 105503/1-105503/4).
Many phenomena observed at the aqueous interface between a surface and a biological entity such as a protein or a whole mammalian cell are far more complicated than merely simple hydrophobic/hydrophobic interactions (Luk, Y.-Y. et al., Chem. Mater. 2005, 17, (19), 4774-4782; Luk, Y.-Y. et al., Langmuir 2000, 16, (24), 9604-9608). For instance, proteins adsorb and mammalian cells adhere to most surfaces that can be both hydrophobic and hydrophilic (Luk, Y.-Y. et al., Langmuir 2000, 16, (24), 9604-9608; Kane, R. et al., Langmuir 2003, 19, (6), 2388-2391). Multiple chemistries and mechanisms have been proposed to control the adsorption of proteins and adhesion of cells on surfaces (Kane, R. S. et al., Langmuir 2003, 19, (6), 2388-2391; Arakawa, T. et al., Seikagaku 1982, 54, (11), 1255-9; Arakawa, T. et al., Biochem. 1982, 21, (25), 6536-44; Arakawa, T. et al., Biochem. 1985, 24, (24), 6756-62). Nature uses sophisticated molecular forces to assemble complex and uniquely folded structures (Berg, J. M. et al., Biochemistry. 6 ed., W.H. Freeman Company: New York, 2006, p 1120). For example, the base stacking and hydrogen bonding of nucleic acids give rise to the structured double-stranded helix of DNA even though water can compete with the hydrogen bonding between the base pairs (Crothers, D. M. et al., Nucleic Acids: Structures, Properties, and Functions. University Science Books: Sausalito, Calif., 2000; p 808). A folded protein excludes most of the water from the interior of a folded structure while maintaining the polar amino acid residues in the active site (Creighton, T., Proteins: Structures and Molecular Properties. 6 ed.; W.H. Freeman Company: New York, 1992; p 512).
Many methods have been developed to immobilize proteins on materials. For example, proteins have been immobilized on agarose (a galactose-based polyssacharide) (Spagna, G., R. N. Barbagallo, P. G. Pifferi, R. M. Blanco, and J. M. Guisan, Stabilization of a beta-glucosidase from Aspergillus niger by binding to an amine agarose gel. J. Mol. Catal. B: Enzym., 2000. 11(2-3): p. 63-69; Axen, R. and S. Ernback, Chemical fixation of enzymes to cyanogen halide activated polysaccharide carriers. European Journal of Biochemistry, 1971. 18(3): p. 351-60; Axen, R., J. Porath, and S. Ernback, Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature, 1967. 214(5095): p. 1302-4; Hearn, M. T. W., 1,1′-Carbonyldiimidazole-mediated immobilization of enzymes and affinity ligands. Methods Enzymol. FIELD Full Journal Title: Methods in Enzymology, 1987. 135(Immobilized Enzymes Cells, Pt. B): p. 102-17; and Wei, Y., G. Ning, H.-Q. Zhang, J.-G. Wu, Y.-H. Wang, and K.-D. Wesche, Microarray preparation based on oxidation of agarose-gel and subsequent enzyme immunoassay. Sens. Actuators, B FIELD Full Journal Title: Sensors and Actuators, B: Chemical, 2004. B98(1): p. 83-91), polyacrylonitrile (PAN) membranes (Biondi, P. A., M. Pace, O. Brenna, and P. G. Pietta, Coupling of enzymes to polyacrylonitrile. Eur. J. Biochem., 1976. 61(1): p. 171-4; Godjevargova, T., R. Nenkova, and V. Konsulov, Immobilization of glucose oxidase by acrylonitrile copolymer coated silica supports. J. Mol. Catal. B: Enzym., 2006. 38(2): p. 59-64; Hicke, H.-G., P. Boehme, M. Becker, H. Schulze, and M. Ulbricht, Immobilization of enzymes onto modified polyacrylonitrile membranes: application of the acyl azide method. J. Appl. Polym. Sci., 1996. 60(8): p. 1147-61; Hunter, M. J. and M. L. Ludwig, The reaction of imidoesters with proteins and related small molecules. J. Am. Chem. Soc., 1962. 84: p. 3491-504), mesoporous silica (Chaudhary, Y. S., S. K. Manna, S. Mazumdar, and D. Khushalani, Protein encapsulation into mesoporous silica hosts. Microporous Mesoporous Mater., 2008. 109(1-3): p. 535-541; Vinu, A., N. Gokulakrishnan, T. Mori, and K. Ariga, Immobilization of biomolecules on mesoporous structured materials. Bio-Inorg. Hybrid Nanomater., 2008: p. 113-157; Slowing, I. I., B. G. Trewyn, and V. S. Y. and F. Caruso, Bioinspired porous hybrid materials via layer-by-layer assembly. Bio-Inorg. Hybrid Nanomater., 2008: p. 209-238) via often multiple steps of chemical reactions. Some conjugation methods including disulfide (Carlsson, J., R. Axen, and T. Unge, Reversible, covalent immobilization of enzymes by thiol-disulfide interchange. Eur. J. Biochem., 1975. 59(2): p. 567-72; Ljungquist, C., B. Jansson, T. Moks, and M. Uhlen, Thiol-directed immobilization of recombinant IgG-binding receptors. Eur. J. Biochem. FIELD Full Journal Title: European Journal of Biochemistry, 1989. 186(3): p. 557-61) and imine (Spagna, G., R. N. Barbagallo, P. G. Pifferi, R. M. Blanco, and J. M. Guisan, Stabilization of a beta-glucosidase from Aspergillus niger by binding to an amine agarose gel. J. Mol. Catal. B: Enzym., 2000. 11(2-3): p. 63-69; Blanco, R. M. and J. M. Guisan, Protecting effect of competitive inhibitors during very intense insolubilized enzyme-activated support multipoint attachments: trypsin (amine)-agarose (aldehyde) system. Enzyme and Microbial Technology, 1988. 10(4): p. 227-32; Shainoff, J. R., Zonal immobilization of proteins. Biochemical and Biophysical Research Communications, 1980. 95(2): p. 690-5) bond formation between protein and solid support are reversible and unstable at certain pH, and thus are less desired methods. The locations of the immobilized protein in materials are often not controlled. Overall, these methods are primarily aimed for biotechnology purposes such as making the enzyme reusable and the product easily purified (Ong, E., J. M. Greenwood, N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, The cellulose-binding domains of cellulases: tools for biotechnology. Trends Biotechnol., 1989. 7(9): p. 239-43). However, there is a need for new ways of immobilizing proteins for a variety of uses.
Relative to the success in the semi-conductor and electronic industry, man-made materials with biological functions aimed for potential artificial cells and organ still fall embarrassingly far behind the powerful and sophisticated biological machinery such as enzymes, DNA transcription, protein synthesis or a whole organ such an eye or a heart. Several novel core-shell and sophisticated porous materials have been made by using colloidal and soft matter sciences (Jiang, P., J. F. Bertone, and V. L. Colvin, A lost-wax approach to monodisperse colloids and their crystals. Science (Washington, D.C., U.S.), 2001. 291(5503): p. 453-457; Utada, A. S., E. Lorenceau, D. R. Link, P. D. Kaplan, H. A. Stone, and D. A. Weitz, Monodisperse Double Emulsions Generated from a Microcapillary Device. Science (Washington, D.C., U.S.), 2005. 308(5721): p. 537-541; Kuykendall, D. W. and S. C. Zimmerman, Nanoparticles: A very versatile nanocapsule. Nat. Nanotechnol., 2007. 2(4): p. 201-202.). Others have used inorganic templates (Caruso, F., R. A. Caruso, and H. Moehwald, Nanoengineering of inorganic and hybrid hollow spheres by colloidal templating. Science, 1998. 282(5391): p. 1111-1114.). These studies have focused on building novel structures for applications such as drug delivery, but not on biocompatibility issues such as how the chemistry of materials influence the immobilized protein activities or how materials can be rationally engineered such that the immobilized protein will have superior catalytic activities. For artificial materials to mimic or surpass the functions in biological systems, materials must be made highly biocompatible for supporting the biological functions from immobilized proteins, and with structures that allow efficient transport of reagents in and out of the materials (FIG. 1).
An “emulsion” is generally known in the art as two immiscible liquids mixed together (by shaking for example) with small droplets of one liquid dispersed (i.e., separated and distributed throughout the space) in the other liquid. This dispersion is usually not stable and all the droplets will “clump” together over time and forms two layers. Because of the immiscibility, the emulsion is classified according to the chemical nature of the liquids. For example, an emulsion may be classified as an oil-in-water (O/W) emulsion (e.g., micelles), a water-in-oil (W/O) emulsion (e.g., inverted micelles), and sometimes such as water-in-oil-in-water (W/O/W). These classical types of emulsions can be stabilized from coalescence (i.e. preventing the droplets from clumping together) by the presence of surfactant molecules, of which part of the molecular structure is soluble in water, and the other part is soluble in oil-like solvents (see FIG. 2A).
One of the most general and paramount challenges for designing surfaces and materials that are in contact with biological fluids and living systems is biofouling at three different levels: protein adsorption (Wilson, C. J., R. E. Clegg, D. I. Leavesley, and M. J. Pearcy, Mediation of Biomaterial-Cell Interactions by Adsorbed Proteins: A Review. Tissue Engineering, 2005. 11(1/2): p. 1-18; Magnani, A., G. Peluso, S. Margarucci, and K. K. Chittur, Protein adsorption and cellular/tissue interactions. Integrated Biomaterials Science, 2002: p. 669-689; Mrksich, M. and G. M. Whitesides, Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells. Annual Review of Biophysics and Biomolecular Structure, 1996. 25: p. 55-78), mammalian cell adhesion (Mrksich, M., L. E. Dike, J. Tien, D. E. Ingber, and G. M. Whitesides, Using microcontact printing to pattern the attachment of mammalian cells to self-assembled monolayers of alkanethiolates on transparent films of gold and silver. Experimental Cell Research, 1997. 235(2): p. 305-313), and biofilm formation (Callow, J. A. and M. E. Callow, Biofilms. Progress in Molecular and Subcellular Biology, 2006. 42(Antifouling Compounds): p. 141-169; Coetser, S. E. and T. E. Cloete, Biofouling and biocorrosion in industrial water systems. Critical Reviews in Microbiology, 2005. 31(4): p. 213-232). These biofoulings cause a wide range of problems for the biomedical research, public health (Parsek, M. and P. Singh, Bacterial Biofilms: An Emerging Link to Disease Pathogenesis. Annual Review of Microbiology, 2003. 57: p. 677-701; Costerton, J. W., P. S. Stewart, and E. P. Greenberg, Bacterial biofilms: a common cause of persistent infections. Science, 1999. 284(5418): p. 1318-1322), and industry (Coetser, S. E. and T. E. Cloete, Biofouling and biocorrosion in industrial water systems. Critical Reviews in Microbiology, 2005. 31(4): p. 213-232; Cloete, T. E., Biofouling control in industrial water systems: what we know and what we need to know. Materials and Corrosion, 2003. 54(7): p. 520-526).
First, proteins adsorb to almost all types of surfaces, significantly compromising the effort to build multi-array protein assays that require proper orientation and non-denatured protein structure in a small area (Lee, Y.-S, and M. Mrksich, Protein chips, from concept to practice. Trends in Biotechnology, 2002. 20(12, Suppl.): p. S14-S18). Protein adsorption and mammalian cell adhesion are also the primary cause of undesired resistance to medical implants that result in inflammation and sometimes life threatening situations (Wilson, C. J., R. E. Clegg, D. I. Leavesley, and M. J. Pearcy, Mediation of Biomaterial-Cell Interactions by Adsorbed Proteins: A Review. Tissue Engineering, 2005. 11(1/2): p. 1-18; Magnani, A., G. Peluso, S. Margarucci, and K. K. Chittur, Protein adsorption and cellular/tissue interactions. Integrated Biomaterials Science, 2002: p. 669-689). Second, under conditions that support proliferation, most types of mammalian cells adhere to surfaces as an essential requirement for vitality. This adhesion also is also a primary source for the undesired immuno-resistance of medical implants (Wilson, C. J., R. E. Clegg, D. I. Leavesley, and M. J. Pearcy, Mediation of Biomaterial-Cell Interactions by Adsorbed Proteins: A Review. Tissue Engineering, 2005. 11(1/2): p. 1-18; Magnani, A., G. Peluso, S. Margarucci, and K. K. Chittur, Protein adsorption and cellular/tissue interactions. Integrated Biomaterials Science, 2002: p. 669-689; Brunette, D. M., Principles of cell behavior on titanium surfaces and their application to implanted devices. Titanium in Medicine, 2001: p. 485-512). Third, through a multi-step process, microbes (bacteria and fungi) form films of multicellular structures imbedded in a sticky polysaccharide matrix that strongly attaches to surfaces (Callow, J. A. and M. E. Callow, Biofilms. Progress in Molecular and Subcellular Biology, 2006. 42(Antifouling Compounds): p. 141-169). These biofilms cause a wide spectrum of health-related problems, including infections due to medical devices such as intravenous catheters, joint prostheses, cardiac pacemakers, prosthetic heart valves, peritoneal dialysis catheters and cerebrospinal fluid shunts (Parsek, M. and P. Singh, Bacterial Biofilms: An Emerging Link to Disease Pathogenesis. Annual Review of Microbiology, 2003. 57: p. 677-701; Costerton, J. W., P. S. Stewart, and E. P. Greenberg, Bacterial biofilms: a common cause of persistent infections. Science, 1999. 284(5418): p. 1318-1322). These biofilms also cause billions of dollars worth of damage in industries including enhanced corrosion of metallic materials and equipments (Coetser, S. E. and T. E. Cloete, Biofouling and biocorrosion in industrial water systems. Critical Reviews in Microbiology, 2005. 31(4): p. 213-232; Cloete, T. E., Biofouling control in industrial water systems: what we know and what we need to know. Materials and Corrosion, 2003. 54(7): p. 520-526).
Two challenges significantly hinder the development of methods to control biofouling. First, the mechanism of biofouling is not well-understood at molecular level. Although both mammalian cell adhesion and biofilm formation proceed with protein adsorption at the early stage of bio fouling, protein adsorption itself is a multi-step and complex process. There are mixed opinions and theories as to how a surface can resist protein adsorption. Thus, this challenge calls for a reevaluation of the current opinions and the development of new theories. It also requires an experimental system that can both test the new hypotheses and potentially achieve competent anti-fouling surfaces. Second, living organisms continuously sense, respond to (at genetic level) and modify their environments. Thus, even a competent anti-fouling surface may be compromised after prolonged exposure to biological fluids.
Thus, there is a need to work beyond the two-dimensional surfaces and consider a porous interfacial structure that can accommodate both chemical and biological changes over time. Further, a need exists for more advanced and efficient means to (i) study the effect of the chemistry of the gel material on enzymatic activity of immobilized proteins and (ii) tailor different microenvironments on the porous hydrogel for enabling the immobilized enzymes to have a higher catalytic activity than that of free enzymes in solution, (iii) provide a new one-pot method using water-in-water emulsion to make protein-immobilized hydrogel and (iv) introduce a hierarchy in the novel structures of hydrogel allowing preferred location of immobilized proteins.
The present invention is useful for overcoming the various deficiencies in the art.